Sturge–Weber syndrome & port-wine stain: Genomic analysis reveals somatic activating mutation in GNAQ as genetic basis for neurocutaneous disorder

Matt Shirley

22 May 2013

Pevsner lab projects

  1. Identify regions and magnitude of structural variation in LCLs:
  2. Determine the genetic basis for Sturge-Weber syndrome

Outline

Pathology of the Sturge-Weber syndrome

How common is Sturge-Weber syndrome?

Mikhail Gorbachev, president of former USSR

Mikhail Gorbachev, president of former USSR

Representative images of subjects from this study

(A,B) Photographs of a subject with the Sturge–Weber syndrome. (C) PWS from subject without SWS. (D,E,F) Brain MRI from subject with SWS. Arrows: white - atrophy, yellow - leptomeninges, red - choroid. Shirley et al. 2013 NEJM

(A,B) Photographs of a subject with the Sturge–Weber syndrome. (C) PWS from subject without SWS. (D,E,F) Brain MRI from subject with SWS. Arrows: white - atrophy, yellow - leptomeninges, red - choroid. Shirley et al. 2013 NEJM

Rudolf Happle's hypothesis: a somatic mutation escaping lethality

Outline

Exploratory sequencing from paired whole genomes

Paired sample collection + >30X whole genome sequencing:

  1. Identify all mutations in a normal sample
  2. Identify all mutations in the affected sample
  3. Combine the two signals, accounting for some level of background noise in the data

For somatic mutation detection from paired samples we use Strelka1.

How do we identify the correct somatic mutation?

Using Strelka, we identified somatic SNVs in each affected tissue

Subject Somatic SNVs Tissue
1 325 PWS
2 543 Brain
3 427 PWS

Functional annotation of variants using VAAST

Variants in → disease alleles out

1294 variants → 1 candidate variant: GNAQ R183Q

Expanding our list of shared somatic variants

Blue box: variants having a mutant allele in affected sample and not in normal tissue samples

M = sum of log transformed allele frequencies
A = difference of log transformed allele frequencies

Set intersection of low-confidence somatic variants still identifies GNAQ

658 somatic variants: total variants (coding variants)
GNAQ is still the only shared coding somatic variant

GNAQ is a G-protein alpha subunit

Image from EuroEPINOMICS3

GNAQ somatic mutations are associated with uveal melanomas and melanocytic lesions

Targeted deep amplicon sequencing design: primers

Custom PCR amplicon sequencing with Illumina MiSeq

Error correction barcodes

in  fixed   checksum
CATAACT CAAAACT A > T at pos 3
AGAGAGA AGAGAGA ok
TCACAGC TCACAGC ok
GAACAGG GAAAAGG A > C at pos 4
CTATAGT CTATAGT ok
TTTAAAN NNNNNNN bad
NNNNNNN NNNNNNN bad

Targeted deep amplicon sequencing design: read depth

1,000,000 draws from an allele pool with 1% mutant, 0.5% threshold for detection

Based on this simulation, we targeted > 10,000 and achieved 2446 to 93,008 (median, 12,947) read depth.

Results: GNAQ somatic mutation is found in PWS and SWS brain

# Subjects Tissue SWS GNAQ R183Q Detection method
9 PWS Yes 100% Amplicon seq
7 Skin Yes 14% Amplicon seq
13 PWS No 92% Amplicon seq, Primer extension
18 Brain Yes 88% Amplicon seq
6 Brain No 0% Amplicon seq
4 Brain CCM 0% Primer extension
669 Blood/LCL N/A 0.7% Exome seq

CCM = cerebral cavernous malformation
Amplicon seq = 12,947X median read depth
Exome seq = 271X median read depth
Primer extension = SNaPshot allele-specific primer extension

Outline

Effect of R183Q GNAQ mutation on MAPK signaling pathways

Outline

GNAQ regulation by Regulator of G-protein signaling (RGS)

RGS4 serves as a GTPase-activating protein for GNAQ

Closed circle: Mutant GNAQ alone, Open circle: Mutant GNAQ plus RGS4

Closed circle: Mutant GNAQ alone, Open circle: Mutant GNAQ plus RGS4

Berman 1996 Cell

GNAQ interaction with Trio (GEF) activates MAPK pathway independent of PLC-beta

Trio mediates GNAQ activation of p38 and JNK8

Two arms of the MAPK pathway

Two arms of the MAPK pathway

Vaqué 2012 Molecular Cell

Conclusions / Implications

Questions?

Acknowledgements

Lab members

Dr. Jonathan Pevsner
Dr. Joseph Baugher
Dr. Eric Stevens
Larry Frelin
Don Freed
Amanda and Genevieve

Thesis committee members

Dr. Garry Cutting
Dr. Dani Fallin
Dr. Sarah Wheelan
Dr. Ingo Ruczinski

Collaborators

Dr. Anne Comi (Kennedy Krieger Institute)
Dr. Douglas Marchuk (Duke University)
Dr. Bernard Cohen (Kennedy Krieger Institute)
Dr. Paula North (Medical College of Wisconsin)
Dr. Hao Tang (Duke University)
Carol Gallione (Duke University)

Patients and families

BCMB program at Hopkins
Dr. Carolyn Machamer
Margie Policastri
Sharon Root
Dr. Leslie Brown
Dr. Arhonda Gogos

Hopkins friends

My family

My family

Our animals

Our growing family


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  2. Yandell, M., Huff, C., Hu, H., Singleton, M., Moore, B., Xing, J., et al. (2011). A probabilistic disease-gene finder for personal genomes. Genome Research, 21(9), 1529–1542. doi:10.1101/gr.123158.111

  3. http://158.64.76.154/euroepinomicsblog/post/1640-sturge-weber-syndrome-explained-somatic-mutations-in-gnaq

  4. Lee, C.-W., Choi, D.-Y., Oh, Y.-G., Yoon, H.-S., & Kim, J.-D. (2005). An infantile case of Sturge-Weber syndrome in association with Klippel-Trenaunay-Weber syndrome and phakomatosis pigmentovascularis. Journal of Korean medical science

  5. Robaee, Al, A., Banka, N., & Alfadley, A. (2004). Phakomatosis pigmentovascularis type IIb associated with Sturge-Weber syndrome. Pediatric Dermatology

  6. Bystrykh, L. V. (2012). Generalized DNA Barcode Desig Based on Hamming Codes. (J.-A. L. Stanton, Ed.)PLoS ONE.

  7. Berman, D. M., Wilkie, T. M., & Gilman, A. G. (1996). GAIP and RGS4 Are GTPase-Activating Proteins for the G Subfamily of G Protein ? Subunits. Cell.

  8. Vaqué, J. P., Dorsam, R. T., Feng, X., Iglesias-Bartolome, R., Forsthoefel, D. J., Chen, Q., et al. (2012). A Genome-wide RNAi Screen Reveals a Trio-Regulated Rho GTPase Circuitry Transducing Mitogenic Signals Initiated by G Protein-Coupled Receptors. Molecular Cell